Differential Oral Microbial Input Determines Two Microbiota Pneumo‐Types Associated with Health Status

Abstract The oral and upper respiratory tracts are closely linked anatomically and physiologically with the lower respiratory tract and lungs, and the influence of oral and upper respiratory microbes on the lung microbiota is increasingly being recognized. However, the ecological process and individual heterogeneity of the oral and upper respiratory tract microbes shaping the lung microbiota remain unclear owing to the lack of controlled analyses with sufficient sample sizes. Here, the microbiomes of saliva, nasal cavity, oropharyngeal area, and bronchoalveolar lavage samples are profiled and the shaping process of multisource microbes on the lung microbiota is measured. It is found that oral and nasal microbial inputs jointly shape the lung microbiota by occupying different ecological niches. It is also observed that the spread of oral microbes to the lungs is heterogeneous, with more oral microbes entering the lungs being associated with decreased lung function and increased lung proinflammatory cytokines. These results depict the external shaping process of lung microbiota and indicate the great value of oral samples, such as saliva, in monitoring and assessing lung microbiota status in clinical settings.


Neutral Model
The fitting of this parameter was performed in R using non-linear least-squares fitting and the minpack.lm package. The goodness of fit of this curve was assessed using the coefficient of determination (R 2 ). Binomial proportion 95% confidence intervals around the model predictions were calculated using the Wilson score interval in the HMisc package in R.

Study recruitment and sample collection
The cohort inclusion criteria for this study are as follows: age > 18 years, no acute respiratory symptoms; not receiving antimicrobial within 3 months before screening or glucocorticoid drug treatment; not immunodeficiency; not pregnant or being breastfeeding. Patients who had taken any antibiotics in the last three months were excluded since antibiotics exert significant effects on the microbiome in the respiratory tract and other sites. Except for lung cancer and three lung cancer patients with bronchiectasis, participants had no other respiratory complications.
The inclusion criteria for lung cancer patients are as follows: lung cancer was diagnosed by histopathology in the lung intensive care unit (PCCM), thoracic surgery and oncology department, without lung infection and other respiratory symptoms. The control participants were volunteered through online registration on the website of the Fifth Affiliated Hospital of Sun Yat-sen University, and were followed up by telephone call and on-site screening to assess the condition of respiratory symptoms and family history of respiratory disease. All participants have fully understood the operation situation and signed the informed consent form. The clinical samples were taken two to three days after admission, during which time the patient received no antibiotics or corticosteroids but blood oxygen monitoring, temperature and blood pressure measurements, and routine in-patient care.
The enrolled 67 lung cancer patients and 32 volunteers without lung disease all underwent bronchoalveolar alveolar lavage fluid, nasal swab, oropharyngeal swab and saliva collection. When bronchoscopy is performed for alveolar lavage, respiratory specialists with an over 5-year experience in bronchoscopy used a nasal approach for research. We avoided suctioning until our scope reaches the position for sampling as possible, as the underlying carry-over of upper airway microorganism to the lung.
Approximately 60 ml of sterile saline in total was passed through the scope channel and about 20-30 ml bronchoalveolar lavage (BAL) was recovered. Samples were immediately frozen at 4 degrees Celsius. Nasal swabs, oropharyngeal swabs, and saliva were collected on the day of bronchoscopy. When collecting nasal swabs and throat swabs, use a sterile cotton swab to rotate the corresponding body part of the subject at least five times. All swabs collected were immediately frozen at -80 degrees celsius. Saliva collection requires subjects to perform before bronchoscopy.

Table S1
The results of pneumo-types in 99 patients. ID is the sample name, SourceTracker group is the grouping after high or low grouping based on saliva contribution value, and DMM group is the grouping based on DMM model.

Table S2
The results of LEfSe and ANCOM.

Table S3
The results of Netshift analysis of salivary microbiota interaction network. N (LOIT/HOIT) is the number of node degrees in LOIT/HOIT, Exclusive is unique in HOIT, DelBet is the delta value from LOIT to HOIT, and COM is the community membership degree of the node in HOIT.

Table S4
Random forest model selects specific taxas at species level, ASV level and genus level.

Table S5
The demographic and clinical data statistics.

Table S6
The results of linear model fitting.

Table S7
The basic statistics of sequencing data.

Table S8
The relative abundance of dominant ASVs in negative controls and clinical samples.